RESUMO
Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2-targeting phosphorylation site-specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2-specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain-only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)9-mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA-mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.-Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C., Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.
Assuntos
Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Animais Recém-Nascidos , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Insuficiência Cardíaca/metabolismo , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
The TIGIT (T cell immunoreceptor with Ig and ITIM domains) protein is a co-inhibitory receptor that has been reported to suppress autoreactive T and B cells to trigger immunological tolerance. We generated a new recombinant protein by connecting the extracellular domain of murine TIGIT to the Fc region of the mouse immunoglobulin IgG2a. The fusion protein was then characterized. The results suggested that among mice with lupus that were treated with the TIGIT-Ig fusion protein, the onset of proteinuria was delayed, serum concentrations of autoantibodies, such as antinuclear antibodies, were reduced without a decrease in the total IgG concentrations, and the survival rate was significantly increased compared to those of the controls. In conclusion, TIGIT-Ig administration showed promising results for both the prevention and treatment of autoimmune diseases in mice. This indicates that treatment with recombinant human TIGIT-Ig shows promise as an effective way to treat human autoimmune diseases.
Assuntos
Imunoterapia/métodos , Lúpus Eritematoso Sistêmico/terapia , Nefrite Lúpica/terapia , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Anticorpos Antinucleares/sangue , Modelos Animais de Doenças , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Camundongos , Camundongos Endogâmicos NZB , Camundongos SCID , Proteínas Recombinantes de Fusão/genéticaRESUMO
Exosome therapy has garnered significant attention due to its natural delivery capabilities, low toxicity, high biocompatibility, and potential for personalised treatment through engineering modifications. Recent studies have highlighted the ability of tumour cell-derived exosomes (TDEs) to interact with immune cells or modify the immune microenvironment to suppress host immune responses, as well as their unique homing ability to parental cells. The core question of this study is whether this immunomodulatory property of TDEs can be utilised for the immunotherapy of inflammatory diseases. In our experiments, we prepared exosomes derived from murine colon cancer cells CT26 (CT26 exo) using ultracentrifugation, characterised them, and conducted proteomic analysis. The therapeutic potential of CT26 exo was evaluated in our dextran sulphate sodium salt (DSS)-induced inflammatory bowel disease (IBD) mouse model. Compared to the control and 293 T exo treatment groups, mice treated with CT26 exo showed a reduction in the disease activity index (DAI) and colon shortening rate, with no noticeable weight loss. Haematoxylin and eosin (H&E) staining of colon paraffin sections revealed reduced inflammatory infiltration and increased epithelial goblet cells in the colons of CT26 exo-treated group. Furthermore, we conducted preliminary mechanistic explorations by examining the phenotyping and function of CD4+ T cells and dendritic cells (DCs) in the colonic lamina propria of mice. The results indicated that the ameliorative effect of CT26 exosomes might be due to their inhibition of pro-inflammatory cytokine secretion by colonic DCs and selective suppression of Th17 cell differentiation in the colon. Additionally, CT26 exo exhibited good biosafety. Our findings propose a novel exosome-based therapeutic approach for IBD and suggest the potential application of TDEs in the treatment of inflammatory diseases.
RESUMO
Cancer immunotherapies rely on one or few specific tumour-associated antigens. However, the adaptive immune system relies on a large and diverse repertoire of antibodies for antigen recognition. Here we report the development and applicability of libraries of immune cells displaying diverse repertoires of chimaeric antigen receptors (CARs) that can recognize non-self antigens and display antigen-dependent clonal expansion, with the expanded population of tumour-specific effector cells leading to long-lasting antitumour responses in mouse models of epithelial tumours. The intravenous injection of synthetic libraries of murine CARs on TET2- T cells led to robust immunological memory and the recognition of mutated or evolved tumours, owing to the maintenance of CAR diversity. Off-the-shelf libraries of 106 murine or human CAR clones displayed on genetically modified human NK-92 cancer cells completely eliminated established tumours in mice with murine xenografts and patient-derived xenografts. Synthetically generated CAR libraries may aid the discovery of new CARs and the development of immunotherapies.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Animais , Antígenos de Neoplasias , Humanos , Imunoterapia , Camundongos , Neoplasias/terapia , Linfócitos TRESUMO
PURPOSE: Both EGFR and PI3K-Akt signaling pathways have been used as therapeutically actionable targets, but resistance is frequently reported. In this report, we show that enrichment of the cancer stem cell (CSC) subsets and dysregulation of Notch signaling underlie the challenges to therapy and describe the development of bispecific antibodies targeting both HER and Notch signaling. EXPERIMENTAL DESIGN: We utilized cell-based models to study Notch signaling in drug-induced CSC expansion. Both cancer cell line models and patient-derived xenograft tumors were used to evaluate the antitumor effects of bispecific antibodies. Cell assays, flow cytometry, qPCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. RESULTS: We found that EGFR/Notch targeting bispecific antibodies exhibited a notable antistem cell effect in both in vitro and in vivo assays. Bispecific antibodies delayed the occurrence of acquired resistance to EGFR inhibitors in triple-negative breast cancer cell line-based models and showed efficacy in patient-derived xenografts. Moreover, the EGFR/Notch bispecific antibody PTG12 in combination with GDC-0941 exerted a stronger antitumor effect than the combined therapy of PI3K inhibitor with EGFR inhibitors or tarextumab in a broad spectrum of epithelial tumors. Mechanistically, bispecific antibody treatment inhibits the stem cell-like subpopulation, reduces tumor-initiating cell frequency, and downregulates the mesenchymal gene expression. CONCLUSIONS: These findings suggest that the coblockade of EGFR and Notch signaling has the potential to increase the response to PI3K inhibition, and PTG12 may gain clinical efficacy when combined with PI3K blockage in cancer treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptores Notch/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Anticorpos Biespecíficos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Receptores ErbB/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Humanos , Indazóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Sulfonamidas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genetically engineered T cells expressing a chimeric antigen receptor (CAR) are rapidly emerging a promising new treatment for haematological and non-haematological malignancies. CAR-T therapy can induce rapid and durable clinical responses but is associated with unique acute toxicities. Moreover, CAR-T cells are vulnerable to immunosuppressive mechanisms. Here, we report that CAR-T cells release extracellular vesicles, mostly in the form of exosomes that carry CAR on their surface. The CAR-containing exosomes express a high level of cytotoxic molecules and inhibit tumour growth. Compared with CAR-T cells, CAR exosomes do not express Programmed cell Death protein 1 (PD1), and their antitumour effect cannot be weakened by recombinant PD-L1 treatment. In a preclinical in vivo model of cytokine release syndrome, the administration of CAR exosomes is relatively safe compared with CAR-T therapy. This study supports the use of exosomes as biomimetic nanovesicles that may be useful in future therapeutic approaches against tumours.
Assuntos
Exossomos/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Exossomos/metabolismo , Humanos , Ativação Linfocitária/imunologia , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Neoplasias/genética , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismoRESUMO
Post-ischaemic heart failure is a major cause of death worldwide. Reperfusion of infarcted heart tissue after myocardial infarction has been an important medical intervention to improve outcomes. However, disturbances in Ca2+ and redox homeostasis at the cellular level caused by ischaemia/reperfusion remain major clinical challenges. In this study, we investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of a Type-2 ryanodine receptor (RyR2) degradation-associated gene, Presenilin 1 (PSEN1), to combat post-ischaemic heart failure. Adeno-associated viral PSEN1 gene delivery elevated PSEN1 protein expression in a post-infarction rat heart failure model, and this administration normalised the contractile dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca2+ handling and function. Moreover, PSEN1 gene transfer to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca2+ leak, thereby restoring the diminished intracellular Ca2+ transients and SR Ca2+ load. Moreover, PSEN1 gene transfer reversed the phosphorylation of RyR2 in failing cardiomyocytes. However, selective autophagy inhibition did not prevent the PSEN1-induced blockade of RyR2 degradation, making the participation of autophagy in PSEN1-associated RyR2 degradation unlikely. Our results established a role of the cardiac expression of PSEN1 with AAV9 vectors as a promising therapeutic approach for post-ischaemic heart failure.
Assuntos
Dependovirus/genética , Vetores Genéticos , Insuficiência Cardíaca/fisiopatologia , Isquemia Miocárdica/genética , Isquemia Miocárdica/fisiopatologia , Presenilina-1/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Homeostase , Ratos , Retículo Sarcoplasmático/metabolismo , Função Ventricular EsquerdaRESUMO
Epidermal growth factor receptor (EGFR) blockade and radiation are efficacious in the treatment of cancer, but resistance is commonly reported. Studies have suggested that dysregulation of Notch signaling and enrichment of the cancer stem cell population underlie these treatment challenges. Our data show that dual targeting of EGFR and Notch2/3 receptors with antibody CT16 not only inhibited signaling mediated by these receptors but also showed a strong anti-stem cell effect both in vitro and in vivo. Treatment with CT16 prevented acquired resistance to EGFR inhibitors and radiation in non-small cell lung cancer (NSCLC) cell line models and patient-derived xenograft tumors. CT16 also had a superior radiosensitizing impact compared with EGFR inhibitors. CT16 in combination with radiation had a larger antitumor effect than the combination of radiation with EGFR inhibitors or tarextumab. Mechanistically, CT16 treatment inhibits the stem cell-like subpopulation, which has a high mesenchymal gene expression and DNA repair activity, and reduces tumor-initiating cell frequency. This finding highlights the capacity of a combined blockade of EGFR and Notch signaling to augment the response to radiation and suggests that CT16 may achieve clinical efficacy when combined with radiation in NSCLC treatment.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Receptores Notch/antagonistas & inibidores , Aldeído Desidrogenase/metabolismo , Animais , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Antineoplásicos/farmacologia , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos SCID , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The human epidermal growth factor receptor (EGFR) targeting chimeric monoclonal antibody, cetuximab (Erbitux®), is a widely used drug in the treatment of metastatic colorectal cancer. However, the activation of the extensive crosstalk among the EGFR family receptors as well as other tyrosine kinase receptors (RTKs) impairs the efficacy of the drug by fueling acquired resistance. To identify the responsible potential activation pathway underlying cetuximab resistance and generate novel treatment strategies, cetuximab-resistant colorectal cancer cell lines were generated and validated and a functional RNAi screen targeting human RTKs was used to identify extensive receptor tyrosine kinase signaling networks established in resistant cancer cells. MET, Axl, and IGF-1R were identified as contributors to the acquired resistance to cetuximab. Targeting vectored immunoprophylaxis (VIPs) to different RTKs were generated and characterized. Different VIP approaches were evaluated in vivo with parental and cetuximab-resistance xenografts and the RTKs in resistant cancer xenografts were inhibited with VIPs via re-sensitization to cetuximab treatment. Combination of VIPs was more broadly efficacious, mechanistically, due to co-blocking the EGFR/Axl/MET signaling pathway, which was cross-activated in the resistant cell lines. Moreover, a VIP-based procedural treatment strategy not only eliminated the tumor but also afforded long-lasting protection from tumor recurrence and resistance. Overall, EGFR-related RTK pathway-network activation represents a novel mechanism underlying cetuximab resistance. A broad VIP combination strategy and VIP-based procedural treatment strategy may be a recommended addition to cetuximab-based targeted therapy. Our results establish a new principle to achieve combined RTK inhibition and reverse drug resistance using a VIP approach.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cetuximab/farmacologia , Neoplasias Colorretais/terapia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Terapia Genética/métodos , Inibidores de Proteínas Quinases/farmacologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Dependovirus/genética , Relação Dose-Resposta a Droga , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/imunologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Interferência de RNA , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
Gold nanoparticles, which have unique physicochemical characteristics, are being used for an increasingly wide range of applications in biomedical research. In this study, gold nanorods (width of 25 nm, length of 52 nm) were found to be internalized by A549 cells and were primarily localized in the lysosomes and membranous vesicles. The integrity of the membranes of A549 cells exposed to gold nanorods for 4h was damaged, as indicated by laser scanning confocal microscopy (LSCM). Increased lactate dehydrogenase (LDH) leakage and decreased cell viability further indicated the concentration-dependent cytotoxicity of the gold nanorods to the A549 cells. Reactive oxygen species (ROS) production was induced in the A549 cells by the gold nanorods, and this effect was positively correlated with the concentration of the gold nanorods. The results of this study indicated that exposure to gold nanorods caused dose-dependent cytotoxicity in A549 cells and that oxidative stress may be the main factor causing cytotoxicity.